Accessibility to residues in transmembrane segment four of the glycine receptor

Glycine receptors (GlyRs) are members of the ligand-gated ion channel superfamily. Each subunit has four transmembrane segments (TM1-TM4). Several studies suggest that amino acids in all four TMs face into a water-filled, alcohol and anesthetic binding cavity in the extracellular portion of the transmembrane domain. TM4 should contribute a "wall" to this cavity, but the residues involved are unknown. Here, we determined the ability of an alcohol analog, propyl methanethiosulfonate (propyl MTS), to covalently react with twelve GlyR TM4 positions (I401-I412) after mutating the original amino acids to cysteines. Reactivity of a cysteine with propyl MTS implies that the cysteine is exposed to water. W407C, I409C, Y410C, and K411C showed altered receptor function following reaction with propyl MTS in the presence or absence of glycine. The cysteine mutations alone eliminated the effects of ethanol for I409C, Y410C, and K411C, and reduced the effects of octanol for I409C and isoflurane for K411C. The ability of propyl MTS to reduce isoflurane and chloroform potentiation was examined in the reactive mutants. Potentiation by isoflurane was significantly reduced for I409C after reaction. These data demonstrate water-accessibility of specific TM4 positions in the GlyR and suggest involvement of these residues with alcohol and anesthetic action.     

Observations on the development of cerebellar afferents in Xenopus laevis

Summary The development of cerebellar afferents has been studied in the clawed toad, Xenopus laevis, from stage 46 to 64, with the horseradish peroxidase retrograde tracer technique. Already in stage 48 tadpoles, i.e. before the formation of the limbs, a distinct set of cerebellar afferents was found. Vestibulocerebellar (mainly arising bilaterally in the nucleus vestibularis caudalis) and contralateral olivocerebellar projections dominate. Secondary trigeminocerebellar (from the descending nucleus of the trigeminal nerve) and reticulocerebellar connections were also found. At stage 50, spinocerebellar projections appear originating from cervical and lower thoracic/upper lumbar levels. The cells of origin of the spinocerebellar projection can be roughly divided in two neuronal types: ipsilaterally projecting large cells, which show a marked resemblance to primary motoneurones (spinal border cells) and smaller contralaterally projecting neurons. Primary spinocerebellar projections from spinal ganglion cells could not be demonstrated.At stage 50, a possible anuran homologue of the mammalian nucleus prepositus hypoglossi was found to project to the cerebellum. In only one of the experiments labeled neurons were found in the contralateral mesencephalic tegmentum. At none of the studied stages a raphecerebellar projection could be demonstrated.It appears that already early in cerebellar development, before the formation of the limbs, most of the cerebellar afferents as found in adult Xenopus laevis are present.     

Early development of descending pathways from the brain stem to the spinal cord in Xenopus laevis

Summary The early development of descending pathways from the brain stem to the spinal cord has been studied in Xenopus laevis tadpoles. The relatively protracted development of this permanently aquatic amphibian as well as its transparency during development make this animal particularly attractive for experimental studies. Between the 5th and 10th myotome the spinal cord was crushed with a thin needle and dry horseradish peroxidase (HRP) crystals were applied. After a survival time of one day the tadpoles were fixed and the brain and spinal cord were stained as a whole according to a modification of the heavy metal intensification of the DAB-reaction, cleared in cedarwood oil and examined as wholemounts.At stage 28 (the neural tube has just closed) the first brain stem neurons projecting to the spinal cord were found in what appear to be the nucleus reticularis inferior and medius. At this stage of development the first, uncoordinated swimming movements can be observed. At stage 30/31 (the tailbud is visible) both Mauthner cells project to the spinal cord as well as the interstitial nucleus of the fasciculus longitudinalis medialis situated in the mesencephalon. Towards stage 35/36 (the tail is now clearly visible), a more extensive reticulospinal innervation of the spinal cord appears, now including cells of the nucleus reticularis superior. At this stage also the first vestibulospinal and raphespinal projections were found. At stage 43/44 (the tadpoles have now a well-developed tail) the pattern of reticulospinal projections appears to be completed with the presence of labeled neutrons in the nucleus reticularis isthmi. From stage 43/44 on, the number of HRP-positive cells is steadily increasing. At stage 47/48, when the hindlimb buds appear, the descending projections to the spinal cord are comparable with the adult situation except for the absence of a rubrospinal and a hypothalamospinal projection.The observations demonstrate that already very early in development reticulospinal fibers and, somewhat later, Mauthner cell axons and vestibulospinal fibers innervate the spinal cord. Furthermore, a caudorostral gradient appears to exist with regard to the development of descending projections to the spinal cord. However, the interstitial nucleus of the fasciculus longitudinalis medialis forms an exception to this rule.     

Immunocytochemical study of glycine receptors in the retina of the frog Xenopus laevis

The expression of glycine receptors in the retina of clawed frog, Xenopus laevis was studied immunocytochemically. Glycine receptors (GlyRs), as revealed by means of several different antibodies, were mainly distributed in the inner (IPL) and the outer plexiform layers. Their composition was determined to include α2 and α3 subunits. Typical punctate appearance and specific lamination in the IPL were seen with each of the antibodies directed against the different GlyRs’ subunits. A notion for diversity of the glycine receptors was put forward, according to which the α2 and α3 subunits are located in different subtypes of glycine synapses.     

Deficient membrane integration of the novel p.N14D-GJB2 mutant associated with non-syndromic hearing impairment

Mutations in GJB2, the gene encoding for the Gap Junction protein Connexin 26 (Cx26), have been established as the major cause of hereditary, non-syndromic hearing impairment (HI). We report here the identification of a novel point mutation in GJB2, c.40A>G [p.N14D], detected in compound heterozygosity with the c.35delG mutation in two brothers with moderate non-syndromic sensorineural HI. The mother who carried one wildtype and a p.N14D allele displayed normal hearing. The mutation leads to substitution of the neutral amino acid asparagine (N) by the negatively charged aspartic acid (D) at amino acid number 14, a position that is conserved among Cx26 of different organisms and among many other connexin isoforms. To investigate the impact of this mutation on protein function, Cx26 activity was measured by depolarization activated hemichannel conductance in non-coupled Xenopus laevis oocytes. Oocytes injected with the p.N14D mutant cRNA showed strongly reduced currents compared to wildtype. Coinjection of wildtype and mutant cRNA at equimolar levels restored the conductive properties supporting the recessive character of this mutation. Total Cx26 protein expression and cell surface abundance examined by western blotting and by quantitative immunoassays revealed that the hemichannel was properly synthesized but not integrated into the plasma membrane. In this study we have shown that the GJB2 mutation p.N14D is associated with recessively inherited HI and exhibits a defective phenotype due to diminished expression at the cell surface.     

Kir2.1/Kir2.3嵌合体的构建与表达

目的构建Kir2.1/Kir2.3通道嵌合体,为进一步研究Kir2.1和Kir2.3通道的调控机制打下基础。方法利用重叠延伸PCR方法构建不同的Kir2.1/Kir2.3嵌合体质粒:N1P3C3,N3P1C1,N3P3C1,N1P1C3,NIP3C1,N3P1C3。将不同的嵌合体质粒分别用NheI线性化后,转录为cRNA表达于非洲爪蟾卵母细胞,用双电极电压钳记录电流。结果不同的Kir2.1/Kir2.3嵌合体质粒构建成功,在非洲爪蟾卵母细胞上可以记录到电流表达。结论成功构建Kir2.1/Kir2.3嵌合体并完成了嵌合体通道的异源性表达和电压钳记录。     

钴对表达在爪蟾卵母细胞上P2X4受体介导的ATP-激活电流的调制

目的 研究钴对P2X4受体介导的ATP-激活电流（ⅠATP）的调制作用.方法 应用双微电极电压钳技术,记录细胞外液加CoCl2对表达在爪蟾卵母细胞上的大鼠颈上神经节P2X4受体介导的ⅠATP的调制作用.结果 0.1 ～30 μmol/L浓度范围的钴离子对P2X4受体介导的ⅠATP呈明显的增强效应,钴浓度越高,ⅠATP幅值越大,0.1μmol/L、0.3μmol/L、1μmol/L、3μmol/L、10 μmol/L、30 μmol/L的钴离子使ⅠATP增加的百分比分别为：12.1±4.6,30.4±5.1,49.3±6.8,70.9±7.2,104.2±16.7,145.8 ±20.1（P ＜0.01）;ⅠATP的量-效曲线在钴离子的作用下向左上移位;应用1 μmol/L钴离子加ATP的混合液同时灌流细胞,可导致ATP的EC50从（15.6±1.9）μmol/L降低到（8.1 ±0.6）μmol/L（P ＜0.01）;即使是高浓度的钴,仍然对ⅠATP表现出明显的增强作用;当ATP浓度不变,钳制电位变化（-140 ～ 60 mV）时,钴离子对P2X4受体介导的ⅠATP的增强作用无明显变化.结论 钴对P2X4受体介导的ⅠATP的增强效应呈浓度依赖性而非电压依赖性.钴与通道蛋白胞外环的作用位点可能与锌和镉不同.     

Imaging CFTR in its native environment

Application of atomic force microscopy (AFM) on isolated plasma membranes is a valuable method to study membrane proteins down to single-molecule level in their native environment. The cystic fibrosis transmembrane conductance regulator (CFTR), a protein of the adenosine triphosphate-binding cassette transporter superfamily, is known to play a crucial role in maintaining the salt and water balance on the epithelium and to influence processes such as cell volume regulation. A mutation in the gene encoding for CFTR results in cystic fibrosis (CF), a very common lethal genetic disease. Identification of CFTR within the cell membrane at the single-molecule level makes it feasible to visualize the distribution and organization of CFTR proteins within the cell membrane of healthy individuals and CF patients. We were able to show that human red blood cells have a CFTR distribution comparable to that of epithelial cells and that the number of CFTR in cells derived from CF patients is strongly reduced. Studies on CFTR-expressing oocytes disclose CFTR dynamics upon CFTR activation. We observed that cyclic adenosine monophosphate induces an insertion of CFTR in the plasma membrane and the formation of heteromeric CFTR-containing structures with yet unknown stoichiometry. The structure of CFTR was identified by high-resolution scans of immunogold-labeled CFTR, revealing that CFTR forms a tail-to-tail dimer with a central pore. In conclusion, these studies show that AFM experiments on isolated plasma membranes allow not only quantification and localization of membrane proteins but also provide insight in their dynamics at a single-molecule level.     

Molecular basis for genistein-induced inhibition of Kir2.3 currents

Inwardly rectifying potassium channels play an important role in the maintenance of membrane potential in neurons and myocardium. Identification of functional regulation mechanisms concerning these channels may lead to the development of specific modulators for these channels. Genistein is an isoflavone with potent inhibitory activity on protein tyrosine kinase. In this study, we have found that among three members of the Kir family (Kir2.3, Kir2.1, and Kir3.4* [a highly active mutant of Kir3.4, Kir3.4-S143T]) we tested, genistein significantly inhibited Kir2.3 currents. Using the two-electrode voltage clamp technique, we have demonstrated that micromole concentrations of genistein concentration-dependently and reversibly inhibited the currents of Kir2.3 channel expressed in Xenopus oocytes with an IC₅₀ of 16.9 ± 2.8 μM. Using the whole-cell patch-clamp technique, genistein also inhibited the currents of Kir2.3 channel expressed in HEK293 cells with an IC₅₀ of 19.3 ± 3.2 μM. Genistein had little or no effect on Kir2.1 and Kir3.4* currents. The effect of genistein on Kir2.3 currents was not affected by vanadate, a potent protein tyrosine phosphatase inhibitor. Furthermore, the effect of genistein was not mimicked by daidzein, an inactive analogue of genistein, or another potent tyrosine kinase inhibitor, tyrphostin 23. Chimeras between Kir2.3 and Kir2.1 channels were constructed to identify molecular basis that distinguished the effect of genistein on these channels. It was found that the transmembrane domains and the pore region of Kir2.3 channel were important determinant for high sensitivity for genistein inhibition.